BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Control of glycolytic flux in directed biosynthesisof uridine-phosphoryl compounds through the manipulationof ATP availability

Yong Chen & Qingguo Liu & Xiaochun Chen & Jinglan Wu &

Jingjing Xie & Ting Guo & Chenjie Zhu & Hanjie Ying

Received: 4 September 2013 /Revised: 14 February 2014 /Accepted: 17 March 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Adenosine triphosphate (ATP), the most importantenergy source for metabolic reactions and pathways, plays avital role in control of metabolic flux. Considering the impor-tance of ATP in regulation of the glycolytic pathway, the useof ATP-oriented manipulation is a rational and efficient routeto regulate metabolic flux. In this paper, a series of efficientATP-oriented regulation methods, such as changing ambienttemperature and altering reduced nicotinamide adenine dinu-cleotide (NADH), was developed. To satisfy the differentdemand for ATP at different phases in directed biosynthesisof uridine-phosphoryl compounds, a multiphase ATP supplyregulation strategy was also used to enhance to yield of targetmetabolites.

Keywords Adenosine triphosphate . Uridine-phosphorylcompounds . Glycolytic pathway . Directed biosynthesis

Introduction

Glycolysis, the most primitive way of obtaining energy, isconsidered the most important metabolic process in the meta-bolic network. Many studies have addressed the fundamentalquestion of what controls the flux during glycolysis, and nu-merous studies have put forward various answers to this ques-tion from different aspects. Much work has focused on theenzyme level of the rate-limiting step in the glycolytic pathway.However, more and more researchers have found that overex-pression, deletion, or introduction of heterologous genes of theglycolytic pathway had no significant effect on the flux of theglycolytic pathway (Müller et al. 1997; Zhou et al. 2008). Thedevelopment of metabolic control theory showed that regula-tion at the level of transcription or translation is of limitedimportance (Somsen et al. 2000). Recently, adenosine triphos-phate (ATP) was found to be responsible for the control ofglycolytic flux. A sudden transition from glucose limitation toglucose excess leads to a new steady state at increased glyco-lytic flux with a sustained decrease in the ATP levels whichsuggested that the ATP level is key in the glycolytic flux(Larsson et al. 1997, 2000). The negative linear correlationbetween ATP level and glycolytic flux is referred to as theATP paradox (Somsen et al. 2000). Subsequent studies con-firmed that the glycolytic flux in Escherichia coli is controlledby the demand for ATP (Koebmann et al. 2002). Using the ATPsupply–demand mode, Aledo et al. (2008) demonstrated thatthe cell is able to sense the energy state, and can either upreg-ulate or downregulate the glycolytic pathway.

Considering the importance of ATP in regulation of theglycolytic pathway, the use of ATP-oriented manipulation is arational and efficient route to regulate metabolic flux.However, one question still remains: How is the ATP levelcontrolled in order to increase the bioprocess efficiency ofATP-based regulation. Regulation of the electron transferchain (ETC), proton gradient, F0F1-ATPase, and oxygen

Chen and Liu contributed equally to this work

Y. Chen :X. Chen : J. Wu : J. Xie : T. Guo :C. Zhu :H. Ying (*)College of Biotechnology and Pharmaceutical Engineering, NanjingUniversity of Technology, Xin mofan Road 5, Nanjing 210009,Chinae-mail: yinghanjie@njut.edu.cn

Y. Chen :Q. Liu : J. Wu : J. Xie : T. Guo : C. Zhu :H. YingNational Engineering Technique Research Center for Biotechnology,Nanjing, China

Y. ChenState Key Laboratory of Motor Vehicle Biofuel Technology,Nanyang, China

X. Chen :H. YingState Key Laboratory of Materials-Oriented Chemical Engineering,Nanjing, China

Appl Microbiol BiotechnolDOI 10.1007/s00253-014-5701-z

supply has been widely used to manipulate intracellular ATPavailability (Zhou et al. 2008). Most research has been fo-cused onmanipulating oxidative phosphorylation, and the roleof substrate-level phosphorylation was wildly underestimated.In fact, respiration is a more efficient pathway in terms of ATPproduction per substrate utilized, but apparently with a limitedcapacity for high rates compared with fermentation (Larssonet al. 1997). Recently, some researchers found a series ofinteresting phenomena, which may provide a new perspectiveon the regulation of substrate-level phosphorylation. Extremechanges were observed in the intracellular concentrations ofadenine nucleotides when Tai et al. (2007) and Postmus et al.(2008) investigated the regulatory mechanisms related toin vivo glycolytic flux through biological systems, and dem-onstrated the dominant role of metabolic control as opposed togene expression in the adaptation of glycolytic enzyme activ-ity to different temperatures. The NADH/NAD+ cofactor pairwas also reported to play a major role in the regulation ofintracellular ATP level (Vemuri et al. 2007). The manipulationof NAD-related metabolic pathways through heterologousexpression of NAD-dependent enzyme (Heux et al. 2006),or supplementation of the culture medium with specific sub-strates for NAD-dependent dehydrogenase, could adjust theintracellular ATP level and significantly alter the glucoseuptake efficiency (Sanchez et al. 2005) and the redistributionof glycolytic flux (Berríos-Rivera et al. 2003a, b, c).

In most bioprocesses, the relationship between glycolyticflux and target metabolites is nonlinear, the extent of ATPdemand differs during the synthesis of target metabolites;therefore, multiphase ATP supply regulation strategies couldbe adopted to enhance the yield of the target metabolites. Weused the metabolism of uridine phosphoryl compounds (UMP,UTP, and UDPG) in Saccharomyces cerevisiae as a modelsystem to investigate how the yeast controlled the glycolyticpathway through ATP regulation. A systems approach wasapplied which involved metabolic flux analysis, enzyme ac-tivity assays, and metabolite analysis. Integrating the informa-tion based on the above analyses, we developed a series ofefficient ATP-oriented regulation methods to satisfy the de-mand for ATP at different phases of synthesis and achieveddirected biosynthesis of uridine phosphoryl compounds.

Materials and methods

Strains, media, and cultivation

The strain used for biosynthesis of uridine phosphoryl com-pounds was S. cerevisiae CY202, a recombinant ofS. cerevisiae CICC 1002, which carries URA5 gene encodingorotate phosphoribosytransferase.

The seed culture medium contained in grams per liter: yeastextract 10, peptone 20, and glucose 20. Medium pH was

adjusted to 5.8 with 1.0MNaOH and 0.1MHCl. Seed cultureswere added at 5 % (v/v) into a 5-l fermenter (NBS Bioflo-110)containing 3 l of fermentation medium, which was in grams perliter: glucose 50, peptone 5, yeast extract 2, (NH4)2HPO4 2,MgSO4·7H2O 1, and KH2PO4 2. Medium pH was adjusted to7.4 as above. Cultivation was at 30 °C for 48 h.

Cells were collected by centrifugation (8,000×g, 10 min at4 °C) and washed twice with distilled water before lyophili-zation and storage at −20 °C.

Biocatalytic reactions

The reaction mixtures of uridine phosphoryl compoundscontained: NaH2PO4 12 g/l, MgCl2 0.4 g/l, dimethylbenzene0.5 % (v/v), and cells (dry cell weight, DCW) 40 g/l.Reactions were carried out in a 5 l fermenter containing 3 lof medium which was kept firstly at room temperature for10min and thenwas added orotic acid 8 g/l and glucose 54 g/l.The pH was adjusted to 8.0 with 1.0 M NaOH.

Reaction mixtures for UTP transformation from UMPcontained: NaH2PO4 9 g/l, MgCl2 0.4 g/l, dimethylbenzene0.5 % (v/v), and cells (DCW) 40 g/l. Reactions were carriedout in a 5 l fermenter containing 3 l of mediumwhich was keptfirstly at room temperature for 10 min and then was addedUMP 11 g/l and glucose 10.8 g/l. The pH was adjusted to 7.0with 1.0 M NaOH.

The reaction mixtures for directed biosynthesis of uridinephosphoryl compounds contained: NaH2PO4 12 g/l, MgCl20.1 g/l, citrate 1 g/l, dimethylbenzene 0.5 % (v/v), and cells(DCW) 40 g/l. Reactions were carried out in a 5 l fermentercontaining 3 l of medium which was kept firstly at roomtemperature for 10 min and then was added orotic acid 8 g/land glucose 54 g/l. Cultivation was carried out at 30 °C for20 h, and the pH was adjusted to 8.0 with 1.0 M NaOH.Supplements to the reaction mixture were 32.4 g glucose,30 ml acetaldehyde, and 27 g NaH2PO4 when the reactiontime reached 20 h. The temperature was adjusted to 37 °C.The reaction mixture was supplemented with 16.2 g glucosewhen the reaction time reached 22 h. The temperature wasadjusted to 20 °C.

Metabolite analysis

ATP was analyzed by the luciferin–luciferase reaction usingthe Entiten ATP assay system FF2000 and detected using themodulus microplate multimode reader. ADP and AMP weremeasured as the difference after enzymatic conversion to ATP.NAD and NADH concentrations were determined as previ-ously described (Du et al. 2006).

Concentrations of UMP, UDP, UTP, and UDPG were mea-sured by high-performance liquid chromatography (HPLC)using a SepaxHP-C18 column (250 mm×4.6 mm×5 μm)and an ultraviolet (UV) detector at 260 nm. The column was

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eluted with 6 % (v/v) phosphoric acid (adjusted to pH 6.6 withtriethylamine) at a flow rate of 1 ml/min at room temperature.

The concentration of glucose was determined using anautomated enzyme analysis system (model SBA-40C).Concentrations of acetate and pyruvate were measured byHPLC with an Aminex HPX-87H column and a UV detectorat 210 nm. The column was eluted with 5 mM sulfuric acid ata flow rate of 0.5 ml/min and 60 °C. The concentration ofethanol and glycerol were measured in yeast culture superna-tants using gas chromatography (Perkin-Elmer GC 8310) witha Poropaq Q-packed column (2 m, 3 mm). The injectorcolumn and flame ionization detectors were set at 210ºC,and N2 was used as a carrier gas at a flow rate of 45 ml/min.Isopropanol was used as an internal standard. The concentra-tions of glucose-6-phosphate (G6P) and 6-phosphogluconate(6PG) were determined by the method according to Sam et al.(1999).

Enzyme assays

Enzyme assays were performed using the modulus microplatemultimode reader at 340 nm with freshly prepared cell ex-tracts. All enzyme activities are expressed as millimole sub-strate converted per minute per milligram protein Units per·milligram protein. In vitro enzyme assays for hexokinase(HXK), phosphofructokinase (PFK), triose phosphate isomer-ase (TPI), and pyruvate kinase (PYK) were performed asdescribed by Mickel and Jansen et al. (2005). 6-phosphoglucose dehydrogenase (GDH) was assayed accord-ing to Zhu and Kazuyuki (2005). Phosphoribosyl diphosphatesynthase (PRS) was assayed according to Arnvig et al. (1990)and UMP kinase (UMPK) was assayed according to Jonget al. (1993). Nucleotide kinase (NDPK) was assayed accord-ing to Jong and Ma (1991). UDPG pyrophosphorylase (UGP)was assayed according to Daran et al. (1995).

Results

Multi-level analysis of temperature-induced altered ATP level,enzyme activity, and glycolytic flux

Changing ambient temperature is the most common environ-mental change that microorganisms have to contend with innature. A systems approach was applied which involved co-factor analysis, enzyme activity assays, and metabolite analy-sis to investigate the response due to temperature stress.Intracellular ATP level was seen to decrease when the tem-perature rose. Compared with values at 37 °C, the ATP levelincreased by 53 % at 30 °C and by 1.2-folds at 20 °C,respectively, while AMP level showed little change at 30 °Cand decreased significantly at 20 °C (Fig. 1a–c). An interest-ing phenomenon found in our experiment was the adenylate

sum (AXP), which was not conserved and decreased withincreased temperature.

The concentrations of most of the measured compoundswere significantly and markedly different. The increased glu-cose consumption rate and the production rate of intermediatemetabolites indicated that the glycolytic rate rose with in-creased temperature (Fig. 2, Table 1), which was consistentwith those reported by Postmus et al. (2012). Combined withthe ATP assay, we found that the glycolytic flux showed astrong negative correlation with intracellular ATP level, whichdemonstrated that ATP exerts some control over the glycolyticpathway. 6PG, an important intermediate metabolite of thepentose phosphate pathway, showed a similar trend to G6P.The optimal temperature for accumulation of uridine phos-phoryl compounds was 30 °C, and the composition of thesecompounds was determined. We found that the optimumtemperatures for accumulation of UMP, UTP, and UDPGwere30, 37, and 20 °C, respectively.

The changes in intermediate metabolites indicated that theenzymes had different responses to temperature (Fig. 3). Togain insight into the effect of temperature on the enzymes, theassays were performed at 20, 30, and 37 °C. Enzymes of theglycolytic pathway, such as HXK, PFK, and PYK, showed asignificant positive correlation with increased temperaturewhen the temperature rose from 20 to 30 °C, while therewas little change between 30 and 37 °C. The activity ofenzymes related to ATP consumption such as PRS, UMPK,and NDK increased 4.3- to 6.3-folds with increased tempera-ture. The optimum temperature for GDH was 30 °C, whichshowed a twofold increase in activity at this temperaturecompared with other temperatures. UGP showed little differ-ence in activity when assayed at different temperatures.

Fig. 1 In vitro adenosine phosphate compounds assays measurement inwhole-cell catalyzed reaction of S. cerevisiae at 20 °C (a), 30 °C (b), and37 °C (c). (■AMP, ●ADP,▲ATP,▼AXP, ◆ATP/AMP). The error barsin the figure indicate the standard derivations from three independentsamples

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Fig. 2 Effect of temperature onglucose consumption (■ 20 °C, ●30 °C,▲ 37 °C). The error bars inthe figure indicate the standardderivations from threeindependent samples

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Decreasing ATP supply through allosteric regulationof substrate phosphorylation in the UMP synthetic process

A sufficient supply of PRPP derived from the pentose phos-phate pathway is necessary for high-level UMP production(Wang et al. 2007). A high ATP supply can suppress theactivity of glucose-6-phosphate dehydrogenase (G6PDH)and introduce too much UTP byproduct (Nahàlka et al.2002). Citrate was added to the culture to regulate the carbon

flux between the glycolytic and pentose phosphate pathways.The lower glucose consumption rate and higher flux of thepentose phosphate pathway (UMP+UTP+UDPG) suggestedthat the substrate-level phosphate had been weakened(Fig. 4a). A decrease in metabolic end products of the glyco-lytic pathway, such as ethanol and acetate, confirmed thispresumption. ATP level was higher when citrate was addedto the medium, which showed a strong negative correlationwith the ATP supply process (Fig. 4b). Our results also

Table 1 In vitro metabolite assays measurement in whole-cell catalyzed reaction of Saccharomyces cerevisiae at 20, 30, and 37 °C

Culture T(degree Celsius)

Time Concentration of metabolite (millimolar)

G6P 6PG FBP Pyr Eth Gly UMP UTP UDPG

20 4 12.9 2.1 5.9 1.4 26.1 3.3 0.8 0 0

8 15.4 2.3 9.7 1.7 82.6 5.4 1.4 0.5 0.3

12 13.9 2.6 9.2 1.9 152.1 8.7 3.8 2.2 1.8

16 8.6 1.7 4.3 1.8 169.6 16.3 8.4 4.0 3.9

20 4.6 1.4 1.9 1.9 165.2 19.6 10.6 3.8 4.8

30 4 16.8 3.3 15.1 1.5 52.2 4.3 2.4 2.1 0

8 10.4 3.1 10.0 2.3 121.7 9.8 4.1 2.7 0.2

12 5.0 2.3 7.0 1.9 234.8 11.9 6.3 3.3 0.5

16 1.8 1.3 0 1.6 230.4 10.1 16.0 4.6 0.8

20 – – 0 1.2 197.8 6.5 24.2 4.4 1.1

37 4 18.6 4.3 22.7 1.8 73.91 7.6 3.3 1.4 0

8 7.5 1.6 6.2 3.1 169.6 13.0 2.9 4.4 0

12 1.4 0.8 0 1.4 230.4 22.8 4.2 8.2 0.3

16 – – – 0.9 178.3 16.9 8.5 13.6 0.5

20 – – – 0.8 134.8 15.6 14.7 8.4 0.7

The data are presented as means from three independent experiments (variation was always <10 %)

Fig. 3 In vitro enzyme assaysmeasurement in whole-cell cata-lyzed reaction of S. cerevisiae at20 °C (black), 30 °C (red), and37 °C (blue). The error bars in thefigure indicate the standard deri-vations from three independentsamples. The error bars in thefigure indicate the standard deri-vations from three independentsamples

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showed that citric acid not only changed the distribution of theflux between the glycolytic pathway and the pentose phos-phate pathway but also led to marked changes in the primaryenergy metabolism of S. cerevisiae. The ethanol pool de-creased by 36 %, while the glycerol pool increased by 49 %following the addition of citrate (Table 2). Another interestingresult was the decreased concentration of acetate, which wasconsidered a consequence of the mismatched glycolytic path-way and TCA cycle.

Manipulating ATP metabolism through the manipulationof NAD availability during UTP synthesis

Manipulation of the redox balance of NADH is considered anefficient strategy for manipulating the intracellular ATP level.Under microaerobic conditions, due to the absence of oxygen,

energy production is mainly from substrate-level phosphory-lation. Continued glycolysis requires that NADH must beoxidized to NAD (Liu et al. 2006). When aldehyde was addedto the medium, intracellular concentrations of NADH andNAD continuously changed. The NAD level increased signif-icantly and resulted in a dramatically altered ratio of NADH toNAD (Fig. 5a). Alteration of intracellular redox balance in-duced a high glucose consumption rate. For the negativecorrelation between glycolytic flux and ATP level, a lowerintracellular ATP level was observed in our experiment(Fig. 5b). Although the level of ATP decreased, the turnoverof ATP increased significantly, which was confirmed by thehigher yield of UTP (Fig. 5c). Another interesting phenome-non was the redistribution of metabolic flux between theglycerol and ethanol pathways. Flux increased through theethanol pathway by 80 %, while flux though the glycerol

Fig. 4 Effect of citrate onglucose, uridine phosphate (a; redline glucose, blue lineUMP, blackline UTP, green line UDPG) andadenosine phosphate (b; red lineAMP, blue line ATP, black lineATP/AMP; ■ control condition, ●5 mM citrate added). The errorbars in the figure indicate thestandard derivations from threeindependent samples

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pathway decreased by 48 % (Table 3). One mole ethanol wasformed with one mole ATP produced, while one mole ATPwas consumed per mole glycerol formed, thus the efficiencyof ATP regeneration also improved significantly.

Directed synthesis of uridine-phosphoryl compoundsthrough multiple-phase ATP supply regulation

It is known that most of the strategies used to enhance theyield of target metabolites with ATP-based manipulation in-clude increasing ATP supply or decreasing ATP supply (Aokiet al. 2005; Blank et al. 2005). However, the biosynthesis ofdifferent uridine-phosphoryl compounds has different ATPrequirements. Multiple-phase ATP supply regulation couldbe developed to achieve a high yield of target metabolites.During the first stage of the reaction, citrate was added to themedium to decrease the ATP supply, which increased themetabolic flux through the pentose phosphate pathway. Theyield of UMP achieved was 52 %, while other uridine-phosphoryl compounds only yielded small proportions, whichwas important for downstream separation. During the secondstage of the reaction, the temperature was adjusted to 37 °C,while acetaldehyde was added to the medium to adjust theredox balance. The regeneration rate of ATP improved signif-icantly, and almost all UMP was transformed into UTP.During the last stage of the reaction, the reaction temperaturewas lowered to restrict ATP availability, which induced fluxredistribution between the glycolytic pathway and the sugarnucleotide pathway. Accumulation of UDPGwas enhanced to16.6 mM (Fig. 6).

Discussion

Uridine-phosphoryl compounds (UMP, UTP, and UDPG),which act as glycosyl donors and contribute to oligosaccha-ride synthesis, are widely used as important pharmaceuticalintermediates and food additives (Dwek 1996). The directedbiosynthesis of uridine-phosphoryl compounds from orotic

acid has many problems, the most important problem beingthe different requirements for flux distribution in the glyco-lytic pathway, pentose phosphate pathway, TCA cycle, andpart of the Leloir pathway in different reaction stages. Recentresearch showed that ATP, which serves as a substrate, prod-uct, activator, or/and inhibitor in metabolic networks (Nelsonand Cox 2004), plays a central role in the distribution and rateof metabolic fluxes (Zhou et al. 2008). However, ATP avail-ability plays a different role in different metabolic pathways,thus ATP-based regulation must be carried out on specificpathways or bioreactions by dedicated strategies. The multi-phase ATP supply regulation strategy could be introduced intothe system to control the production and release of energy tomeet the different ATP requirements. The regeneration of ATPin microorganisms occurs in two ways, substrate-level phos-phorylation and oxidative phosphorylation. In microaerobicconditions, most ATP production comes from substrate-levelphosphorylation. Temperature, NADH availability, and anallosteric effector may be regulatory candidates for manipu-lating intracellular ATP availability.

Much of the current knowledge on temperature adaptationhas been derived from studies on hot and cold shock, and it isbelieved that gene expression plays a dominant role in regu-lation of the glycolytic pathway (Schade et al. 2004; Kandroret al. 2004). However, recent research demonstrated that met-abolic control played a dominant role rather than gene expres-sion in the adaptation of glycolytic enzyme activity to differ-ent temperatures. It was found that glycolytic flux was closelyassociated with the changes in temperature and showed anegative linear correlation with intracellular ATP level (Taiet al. 2007; Postmus et al. 2008; Wouters et al. 2000).However, this research was focused on the description ofATP during resistance to different temperatures and did notprovide an explanation for the observed ATP changes.Metabolic control theory postulates that glycolytic flux con-trol may occur outside the pathway, for instance, in the pro-cesses that consume the ATP generated during glycolysis(Koebmann et al. 2002). Intracellular ATP level is determinedby the rate of the ATP-supplying process in combination with

Table 2 In vitro metabolite as-says measurement in whole-cellcatalyzed reaction of Saccharo-myces cerevisiae

The data are presented as meansfrom three independent experi-ments (variation was always<10 %)

A control condition, B 5 mM cit-rate added

Time (hour) Metabolite concentration in glycolytic pathway (millimolar)

G6P 6PG Ethanol Glycerol Acetate

A B A B A B A B A B

0 0 0 0 0 0 0 0 0 0

4 16.8 34.8 3.9 7.4 52.1 34.9 3.7 3.6 6.1 4.2

8 10.4 25.7 3.6 6.9 127.3 71.8 8.9 9.4 12.8 7.3

12 5.0 15.9 2.7 5.6 242.0 147.0 12.9 15.4 21.4 13.6

16 1.8 4.3 1.5 1.7 234.5 156.9 16.4 21.5 32.7 17.2

20 0.7 2.2 0.9 0.9 209.3 146.4 7.9 24.5 42.1 20.2

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Fig. 5 Effect of acetaldehyde oncofactor (a; red line NADH, blueline NAD, and black lineNADH/NAD), adenosine phos-phate compounds (b; red lineAMP, blue line ATP, and blackline ATP/AMP), and uridinephosphate compounds (c; red lineUMP, blue line UDP, and blackline UTP; ■ control condition, ●10 mM acetaldehyde added). Theerror bars in the figure indicate thestandard derivations from threeindependent samples

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the ATP-consuming process. A rational interpretation for thechanges in ATP could be that the ATP-consuming process ismore sensitive to temperature than the ATP-supplying pro-cess. To address the issue of whether ATP consumption in-duced by temperature change determines the steady-state fluxthrough glycolysis, the processes that consume the ATP gen-erated during glycolysis (the ATP demand) should be investi-gated. As a result of the method of whole-cell transformation,the influence of vertical regulation on the enzyme activitycould be ignored. Temperature has a marked effect on thecatalytic properties of enzymes, however, each enzyme hasunique catalytic properties, including an optimum tempera-ture. PDS, UMPK, and NDK showed significantly highercatalytic activity with increased temperature, which demon-strated an increase in the ATP-consuming rate. The remark-able increase in flux throughout ATP-consuming reactionsinduced a decrease in intracellular ATP level. This does notmean that overall ATP consumption increased more than itsproduction. In fact, ATP consumption and production were

equal. When the ATP consumption rate is stimulated, the ATPlevel can be decreased to maintain a new steady state. Therewas a strong negative correlation between the ATP level andglycolytic flux which would be the main reason for highglycolytic flux at higher temperatures. However, the responseto temperature was different for each enzyme, which inducedflux redistribution in the glycolytic pathway, pentose phos-phate pathway, and Leloir pathway and a significantly alteredspectrum of uridine-phosphoryl compounds. From a microbi-al physiology perspective, the metabolic regulation inducedby altered temperature seems logical. The pentose phosphatepathway is believed to be the major source of NADPH andribose required for biosynthetic reactions and cell growth.This would account for the optimum temperature during theproduction of UMP being similar to the optimum temperaturefor growth. UDPG is the starting point for the synthesis oftrehalose 6-phosphate and glycogen, which are importantreserve carbohydrates. The accumulation of UDPG indicatedthat the glycolytic capacity was saturated, and it was a type of

Table 3 In vitro glucose uptakeand metabolite assays measure-ment in whole-cell catalyzed re-action of Saccharomycescerevisiae

The data are presented as meansfrom three independent experi-ments (variation was always<10 %)

A control condition, B 10 mM ac-etaldehyde added

Time (minute) Metabolite concentration in glycolytic pathway (mM)

Glucose G6P 6PG Ethanol Glycerol Acetate

A B A B A B A B A B A B

0 60 60 0 0 0 0 0 0 0 0 0 0

10 43 38 7.4 8.7 2.4 3.5 3.0 11.4 1.3 0 0 1.3

20 27 13 14.2 15.4 6.0 5.9 14.2 28.3 5.1 2.9 0.5 4.3

40 16 0 19.3 16.9 4.1 3.7 28.4 46.3 14.1 5.4 2.1 9.1

60 2 0 17.4 17.1 3.2 2.7 32.1 56.6 17.2 8.6 4.4 11.2

80 0 0 15.5 15.2 2.6 2.4 36.3 63.1 21.0 13.9 7.2 12.4

Fig. 6 Directed synthesis ofuridine-phosphoryl compoundsthrough multiple-phase ATP sup-ply regulation (● glucose, ▲UMP, ■ UTP, ▼ UDPG). Theerror bars in the figure indicate thestandard derivations from threeindependent samples

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compensation for the reduction in enzyme activity at lowtemperature. Higher temperatures result in higher mainte-nance requirements, thus cells would have to increase theATP supply process, which is beneficial in the accumulationof UTP.

Although cells adjust their flux distribution to adapt toenvironmental temperature changes, they are still not able tomeet the requirements for UMP production.Manymetabolitesfrom the glycolytic pathway and the TCA cycle, such asethanol, glycerol, acetate, malate, and succinate, were detectedin the reaction mixture during the process of UMP production(Chen et al. 2010). More carbon sources could be metabolizedinto the pentose phosphate pathway. Rational flux distributionbetween the glycolytic pathway and the pentose phosphatepathway is necessary for high-level production of UMP. Mg2+

is an essential cofactor of the ATP-dependent enzymes in theglycolytic pathway (Dombek and Ingram 1986, 1987). TheMg-ATP complex, which is a biologically active form forphosphorylation, can be formed by Mg2+ and ATP. Citrateinhibits enzymes by chelating divalent metal ions, causingdepletion of ions in culture (Goel et al. 1999); therefore,cometabolism of citrate and glucose could dramatically reducecarbon flux via the glycolytic pathway. In microaerobic con-ditions, most ATP production was from substrate-level phos-phorylation. Decreased glycolytic flux induced lower ATPavailability. Our research demonstrated that target metaboliteproduction can benefit from a reduction in ATP supply, be-cause lower ATP supply can reduce the formation of by-products such as UTP and UDPG. As previously described,ATP level showed a strong negative correlation with glyco-lytic flux, and the ATP level was at a relatively high levelcompared to that of the control condition. A higher ATP levelcould further inhibit phosphofructokinase and cause phos-phorylated metabolite pools (glucose-6-phosphate and 6-phosphogluconolactone) to rise, which would facilitate thesupply of more precursor metabolites such as 5-phosphoribose and 5-phosphoribose pyrophosphate used forUMP synthesis. A high ATP level could also decrease theactivity of pyruvate kinase, create a bottleneck between theglycolytic pathway and the TCA cycle, weaken the effect ofcarbon overflow, and induce a decrease in organic acids.

A higher UMP concentration could be achieved bydecreasing ATP supply. However, if our target metaboliteis UTP, how could we further increase the target metaboliteconcentration by the regulation of ATP availability?Improving ATP availability was considered an effectivestrategy to enhance the entire process because a largeamount of ATP is needed when UMP is transformed intoUTP. Although high temperature induced a significantlyincreased glycolytic flux in S. cerevisiae, it was still notable to meet the demand for ATP during the process ofUTP production. Here, we manipulated NAD availability toincrease ATP supply.

Under microaerobic conditions, energy production is main-ly from substrate-level phosphorylation. It is of crucial impor-tance for higher glycolytic flux that NADmust be regeneratedconstantly (Bakker et al. 2000; Van Dijken and Scheffers1986). Aldehyde as an exogenous electron acceptor wasadded to the medium to manipulate NAD availability toincrease ATP regeneration capacity which has been repeatedlyemphasized by Yao et al. (2011). A marked decrease in theconcentration of NADH and the ratio of NADH/NAD wasobserved in our experiment. Alteration of intracellular redoxbalance induced increased glycolytic flux and a significantlyaltered spectrum of metabolic products. Metabolic flux anal-ysis showed that the carbon flux via pyruvate node increaseddramatically, while the flux to glycerol decreased. The forma-tion of ethanol and glycerol in yeasts is required to maintainthe redox balance of NADH/NAD. As we know, pyruvatekinase is the rate-limiting step in the glycolytic pathway;therefore, the metabolic rate of ethanol is slower than thereaction catalyzed by glyceraldehyde 3-phosphate dehydroge-nase. To maintain the redox balance, the yeast would have toincrease its glycerol production. Ethanol was the primarymetabolite with the net generation of ATP, and glycerol wasthe primary metabolite with the net consumption of ATP.When analyzing these results by looking at the moles ofATP available per mole of glucose consumed, we found thatnot only the ATP regeneration rate but also the syntheticefficiency of ATP improved significantly. Therefore, manipu-lating the availability of NAD may be an efficient strategy foradjusting intracellular ATP metabolism, which would then bebeneficial for UTP production.

Assessing the effects of altering ATP supply and demandon global metabolic networks, a multiphase ATP supply reg-ulation strategy was established, including temperature con-trol, allosteric regulation, and altering NADH availability.Through ATP-oriented bioprocess optimization, the variedATP demand during the synthesis of target metabolites wassatisfied, and directed biosynthesis of uridine-phosphorylcompounds was achieved. Our work provides a new perspec-tive for achieving regulation of cell physiology and enhancesthe concentration, the yield, and the productivity of targetmetabolites.

Acknowledgments This work was supported by the National Out-standing Youth Foundation of China (Grant no. 21025625), the NationalHigh-Tech Research and Development Program of China (863) (Grantno. 2012AA021200), the National Basic Research Program of China(973) (Grant no. 2011CBA00806), the National Key Technology R&DProgram (Grant no. 2012BAI44G01), the Program Changjiang Scholarsand Innovative in University (Grant no. IRT1066), the National NaturalScience Foundation of China, Youth Program (Grant no. 21106070), theJiangsu Provincial Natural Science Foundation of China (Grant no. SBK201150207), the Priority Academic Program Development (PAPD) ofJiangsu Higher Education Institutions, and the State Key Laboratory ofMotor Vehicle Biofuel Technology (Grant no. 2013003).

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